• Our Behavioral Assays

    The Animal Behavioral Core offers PIs at Cincinnati Children’s and collaborators at other research institutions access to a wide range of behavioral and nonbehavioral assays. The procedures are available for mice or rats, with test apparatus scaled accordingly. Many of our measurement protocols incorporate video tracking and photocell technology, and we use SAS programs to analyze and present the captured data.

    The following tabs describe the assays we offer. For more information, or to arrange a collaboration with the Animal Behavioral Core, contact us at 513-636-8622.

  • Available Assays

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    Acoustic and tactile startle response apparatus.The Acoustic (ASR) and Tactile Startle Response (TSR) are well-established tests for evaluating the complex brainstem-mediated reflex pathway. We use the San Diego Instruments SR Apparatus for these tests.

    We also test for pre-pulse inhibition (PPI), a well-established measure for testing sensorimotor gating. PPI is abnormal in several human disorders, such as schizophrenia and major depression. ASR and PPI test methods and responses are nearly identical in humans and rodents, offering homologous comparisons across species.

    Image of Active Avoidance apparatus.Active/two-way/shuttle box avoidance test using the San Diego Instruments two-sided Gemini System to test the ability of animals to learn to respond to a warning signal (light, tone, or both) that the grid floor is about to deliver a mild aversive stimulus (foot shock). If the animal crosses to the opposite side from where they start, they avoid the shock; if they wait until the warning period is over, the shock is activated and they must escape to the opposite side. Two-way active avoidance is a classically conditioned response where the signal is the conditioned stimulus (CS), the shock is the unconditioned stimulus (US), escape is similar to an unconditioned response (UR), and avoidance is a conditioned response (CR). A typical test requires 20-25 trials/day for 5 days to obtain adequate learning.

    This test can be modified to fit a PI’s specific needs. Typically, the test involves familiarizing the test animal with a test chamber, and then observing changes to the animal’s behavior after it is injected with one of a variety of drug challenges. We typically test animals using indirect dopaminergic agonists, specific dopamine D1 and D2 agonists and antagonists, cholinergic-muscarinic antagonists and glutamatergic-NMDA receptor antagonists. We also have experience using a variety of other agents for this test.

    This uses a labyrinthine maze with ten T-shaped cul-de-sacs branching from a central and circuitous channel leading from the start to the goal. The test is conducted in complete darkness (monitored with an infrared light emitter and camera) to eliminate distal cues, thus testing the animal’s “sense of direction” or ability to navigate using internal, route-based egocentric cues of self-movement (speed and direction).

    We record latency to find the escape and errors of commission (entry into blind alleys). Recent studies we have conducted on this test demonstrate that it depends on neostriatal dopamine but not prefrontal cortex dopamine. The test also is affected by dopamine in the nucleus accumbens but these are accompanied by changes in swimming performance in addition to learning per se and appear to arise from an interaction of motivation and associative factors.

    Image of the Cincinnati Water Maze.

    Image of the Conditioned Fear apparatus.This test uses cued conditioning to evaluate amygdala function and contextual conditioning to evaluate hippocampal function Animals are first conditioned by pairing a neutral stimulus (a tone) to an aversive stimulus (mild footshock) in a specific environment. The animal will associate the tone with the aversive stimulus. A day later, the animal is placed back in the same test environment a second time and observed for reactions to the familiar surrounding with no tone present. Changes in the animal’s reaction reflect hippocampal-dependent changes. Finally, on a third day, the animal is placed in a new, unfamiliar test chamber and given time to explore. After exploration, the tone is presented but no shock is given. Changes in behavior after the tone is presented reflect amygdala-dependent changes.

    Photo of the Elevated Zero Maze.These tests use elevated plus (EPM) or elevated zero (EZP) mazes to test anxiety-like behavior. The mazes are similar, with two open and two  closed  arms or quadrants (closed sections have high, black acrylic walls). Animals are tested for five minutes, and the time spent in the open areas provides the principal index of anxiety. These tests are video tracked and computer scored using AnyMaze software from Stoelting Instruments.

    Image of the Forced Swim Test apparatus.The Forced Swim Test (FST) is a well-known test for assessing stress responses including HPA axis markers such as ACTH, corticosterone, and others. It also serves as a test of depression (the Persolt test of swimming despair). The Persolt test evaluates swimming immobility after a training session in which the animal is placed in a cylinder half-filled with water.

    Image of the Inhibitory/Passive Avoidance apparatus.

    Passive avoidance uses the two-sided Gemini apparatus from San Diego Instruments. For this test, one side is lighted and the other side dark with a door in between. The animal is placed in the lighted side for a period of time, then the door is opened, and the animal is timed for how long it takes them to cross to the dark side. Once they cross to the dark side, the door is closed and they are given a moderate, short, fixed-duration foot shock. They are then removed and tested for retention at different intervals after the first trial to see how long it takes them the second time to reenter the dark compartment. In some procedures trials are repeated until they learn to remain on the lighted side for 3 min whereas in 1-trial passive avoidance they are given only one training trail and retention is tested at some predetermined interval later to see how well they made the dark-shock association. This test will show deficits in animals with hippocampal lesions if the lesion is of sufficient severity.

    Image of the Light-Dark Box apparatus.This test evaluates the time it takes the test animal to move from light to dark areas in a divided test chamber, and dwell time between the two chambers. The test uses our photocell-equipped locomotor activity test chambers.

    Image of Locomotor Activity apparatus.This fully automated test is conducted for various time intervals, but typically lasts 60 minutes. The system records horizontal activity, total distance traveled (cm), central versus peripheral movement or time, vertical movement, repetitive movements and time the test animal spends exploring different zones of the environment. The test measures exploration in response to a novel environment, habituation rate (as the environment become familiar) and baseline spontaneous ambulation. The locomotor activity test can provide data on the test animal’s basal ganglia (caudate-putamen) function.

    Image of Morris Water Maze apparatus.This is the most widely used test for measuring spatial navigation and reference memory. The test animal is placed in an open, circular pool in which there is a submerged platform. A series of tests can measure a variety of memory and navigation tasks, as the test animal swims to the platform from various locations, or tries to find the platform after it has been removed. Tests are video-tracked using AnyMaze, and analysis can provide information on the test animal’s hippocampus-dependent reference memory and prefrontal cortex working memory.

    Image of the Novel Object Recognition apparatus.This test uses the animal’s reaction to a novel object as an avenue to evaluate dorsal hippocampal function. The animal is familiarized with two or more objects, and then is placed in a test space with one of the familiar objects changed to a novel object. We measure time spent exploring the novel object, that if sufficiently preferred by the animal is a reflection of intact memory for the original objects.

    Image of Object Burying apparatus.This test places a grid of foreign objects (marbles) in a standard cage filled with bedding (measured to a standard depth). Animals are tested for 30 minutes, and the number of objects buried is recorded. This is a test of defensive anxiety.

    Image of the Open Space Anxiety Test apparatus.This test is under development and not yet available. The test involves placing animals on an elevated open platform with slanted panels on two sides. The animal is tempted to climb down these slanted surfaces to reach the floor and escape. The slanted panels are designed so the animal can grip but because of the severity of the slope induces fear that it may fall. Consequently, the animal spends most of its time at the edge trying to climb down. This is thought to maintain the animal in an extended state of anxiety. The degree of anxiety is inferred by measuring how much time it spends at the edge versus retreating from the edge to the center of the platform. A reported advantage of this test is that animals can be assessed repeatedly unlike other tests of anxiety that rely on fear of a novel environment which is only applicable once. If the test is validated by the Core it will be made available later in 2016.
    Image of the Radial-Arm Water Maze apparatus.The RWM is a swimming version of the radial-arm maze (RAM) that uses food reward. The Core has an 8-arm RWM. The RWM, like the RAM, tests either spatial trial-dependent, working memory or a combination of spatial working and trial-independent reference memory. In the classic RAM, rats must be food restricted and trained to eat some preferred food reward, then trained to find these rewards or baits within the maze until they learn to search all the arms and go to the very end of an arm to find the bait. After training, testing begins. For working memory, all arms are baited for each new trial. The animal must find all 8 rewards without repeating an arm visit from which it already took one of the rewards. The trial continues until all 8 rewards are retrieved or up to a maximum of 10 min. In the mixed working/reference memory version some arms are baited anew each day and some are never baited. In this version the animal’s task it to find the new rewards each day without repeating an arm entry and never enter the unbaited arms. Our version is a modified version of this that avoids food deprivation and training. One arm in the RWM is always the start and at the end of the other 7 arms is a hidden, submerged platform. On the first trial the animal can enter any arm and reach a platform upon which it is removed and that platform is removed so that on the next trial there are only 6 remaining platforms. The animal’s task is to find any other platform without reentering the one it found first. This continues for 7 trials until each one is found and each reentry to an arm without a platform is counted as an error. The task can be modified to be a mixed working/reference spatial memory task just as the RAM is. The working memory version typically requires 10-14 days for animals to become proficient with the task.
    Image of Rotor-Rod System apparatus.The apparatus is the San Diego Instruments automated rotating rod (or Rotorod) System. The apparatus consists of a rotating drum whose speed is computer controlled. Animals are first taught to walk on the surface at a slow speed. They are then tested in a series of trials in which the speed is increased at regular short intervals until they cannot keep up and fall to a padded surface. How long and at what speed they remain on the drum is a measure of motor coordination and to a lesser extent of balance.
    Image of Social Preference apparatus.This test places a test animal in a three-compartment apparatus with connections between compartments. The two end compartments each contain a small cage with wide slits so that the test animal can smell a conspecific inside the small cage. After familiarization with the apparatus, the test animal is placed in the apparatus with a “stranger” mouse or rat confined to one of the small cages inside one of the end compartments. The test subject is placed in the middle compartment and allowed to explore freely. The time the test subject spends with Stranger-1 versus time in the opposite end compartment with the empty cage reflects social preference. Later, the test subject is placed back in the center compartment but this time a second new animal, Stranger-2, is placed in the small cage that was empty before. How the test animal divides its time between Stranger-1 and Stranger-2 is measured. This test is hypothesized to reflect sociability and social novelty and used as a marker for abnormal social interactions as in Autism Spectrum Disorder (ASD). Most of the data available come from mouse studies in which candidate genes associated with ASD have been modified. How general this test is and how well it predicts human sociability remains to be determined.
    Image of Star Water Maze apparatus.The original Star Maze has 5 arms. Its purpose is to test which learning strategy and type of memory an animal uses to find a hidden platform. Animals are first trained to swim from one specific arm to another where a hidden platform is located. Animals are trained over repeated trials until their performance is nearly flawless. Then they are given a test or probe trial in which they are started from a different arm. If they are spatially navigating, then the start location will not matter and they will swim to the arm that is closest to where the distal cues are that they used to find the platform during training. If they are using internal path cues (egocentric navigation) they will use the same series of right and left turns they used during training and end up in a different (but predictable) location. If they use cues on the walls of different designs, they will end up in yet a different arm. We have adapted the 5-arm design to an 8-arm but the concepts are the same.

    The test measures immobility in the animal by suspending it by the tail using an acrylic plate with a hole for the tail to pass through. Once the animal determines that escape is impossible, it stops struggling. This test can show the effect of antidepressants and sedatives on the animal, and is often used to provide converging evidence for results from Forced Swim Testing (FST). Animals are suspended for single trials of no more than six minutes, and the time spent immobile is manually recorded. This test is available only for use with mice.